Evaluation of Microplastics and Microcystin-LR Effect for Asian Clams (Corbicula fluminea) by a Metabolomics Approach

Microplastics (MP) and microcystins (MCs) are two co-occurring pollutants in freshwater ecosystems that pose significant risks to aquatic organisms and human health. This study investigates the interactions between MP and MCs and their effects on the metabolic responses of freshwater aquaculture. Asian clams have been used as an indicator of microplastic pollution in freshwater ecosystems. The present study investigates metabolic responses of Asian clams during microplastic and microcystin-LR stress to identify health impacts and elucidate mechanistic effects of external stressors on Asian clams. A liquid chromatography/mass spectrometry (LC–MS)-based metabolomics approach was used to identify metabolic perturbations and histological section technique was used to assess changes of tissues from different Asian clam treatment groups. The results showed significantly pathological changes in the gills and hepatopancreas in experimental clam compared to control (healthy) clam. Metabolomics revealed alterations of many metabolites in the hepatopancreas of six Asian clam comparison groups, reflecting perturbations in several molecular pathways, including energy metabolism, amino acid metabolism, protein degradation/tissue damage, and oxidative stress. Overall, this study emphasizes the importance of understanding the interactions between MP and MCs and the need for proactive measures to safeguard freshwater ecosystems and human health.


Introduction
In the past five decades, the demand for plastics has been drastically increasing because of their cost-effective and fine performance, and annual global plastic production has reached approximately 370 million tons in 2019 (Association of Plastic Manufacturers (Organization) 2020; Silva et al. 2020).Polystyrene (PS) plastics are one of the commonly produced plastics and are widely used in various products, such as single-use food packaging, electrical and electronic equipment, building insulation, and inner liner for fridges (Association of Plastic Manufacturers (Organization) 2020).However, plastic pollution becomes a planetary threat considering the large gap in plastic production surge and low reuse rate (Borrelle et al. 2020).Microplastics, which originate from direct manufacturing or the fragmentation and weathering of large plastics, adversely affect every marine and freshwater ecosystem (Borrelle et al. 2020;Huang et al. 2021;Li et al. 2021a).Compared with the marine environment, freshwater ecosystem is closer to human beings (Eerkes-Medrano et al. 2015).Microplastics could be transmitted via the food chain and cause biomagnification, threatening human health (da Costa Araújo and Malafaia 2021).
Over decades, explosive growth of cyanobacteria in freshwaters has led to frequent harmful algal blooms worldwide, which deteriorate water quality by eliciting hypoxia and organism death and cause numerous ecological/economic losses (Qin et al. 2010;Hamilton et al. 2013;Huisman et al. 2018).Microcystis is the most prevalent bloom-forming cyanobacterium in freshwaters and known to produce microcystins (MCs) that synthesized intracellularly.Once released, MCs are toxic to aquatic life and further threaten ecological and human health, thus posing serious eco-risks (Jiang et al. 2011;Li et al. 2017a;Gavrilović et al. 2020;Wan et al. 2021).MC content in freshwaters frequently far exceeds acceptable threshold (1 μg/L) issued by the World Health Organization (Botha et al. 2019;Li et al. 2021b).
Microplastics are considered as a pathway of aquatic products borne hazards due to its capability to act as a vector of compounds that could potentially hamper aquatic products safety.This includes microbial pathogens, heavy metals, and persistent organic pollutants (e.g., microcystin) along with additives, pigments, and dyes intrinsically present in plastics (Barboza et al. 2018).The high surface-to-volume ratio and the hydrophobic polar surface of microplastics facilitate colonization of microbes on microplastic surfaces called plastisphere (Garrido Gamarro et al. 2020).The risk from microplastics to an organism, including human beings, is thus a cumulative effect of exogenous stress exerted by microplastics along with pathogenic effect, leachates of chemical contaminants, and adsorbed heavy metals on it (Wright and Kelly 2017).
The Asian clam (Corbicula fluminea (Müller, 1774)), a typical benthic organism, is one of the widespread and abundant macroinvertebrates established in freshwater benthic ecosystems (Sousa et al. 2008).Being a commercial target species, if contaminated, it has potential implication to human health.A high frequency of microplastic pollution in C. fluminea was already observed in the wild (Su et al. 2018).Furthermore, because of distribution pattern and tolerance to pollutions, C. fluminea has been considered an excellent model for studying the toxicity of numerous pollutants, including heavy metals, persistent organic pollutants, and microplastics (Guo and Feng 2018;Rodrigues et al. 2019;Wang et al. 2021).Studies found the feeding preference of microplastics by C. fluminea and found that the uptake of microplastics varied based on physicochemical properties (Li et al. 2019).Fitness reduction and oxidative lipid damage were observed exposure to microplastics (Oliveira et al. 2018).
In this study, we aimed to investigate the potential impact of microplastics and microcystin-LR on the antioxidant system of C. fluminea and evaluate any associated pathological changes.Mature and healthy individuals were exposed to these pollutants for a duration of 96 h, and the pathological changes in the hepatopancreas, feet, and gill tissues were assessed at five time points (0 h, 24 h, 48 h,72 h, and 96 h).By examining these indicators, we aimed to reveal any acute effects of microplastics and microcystin-LR on C. fluminea.The findings of this study will provide valuable insights into the potential risks of microplastics and microcystin-LR on benthic animals, thus contributing to the development of effective evaluation methods for such pollutants on aquatic organisms.

Experimental Samples
The Asian clams used in this study were sourced from Shanghai, China, with an average weight of 6.75 ± 0.22 g and shell length of 25.28 ± 1.09 mm.Prior to experimentation, the clams were acclimated in a laboratory incubator at a freshwater temperature of 24-25 °C for one week to ensure optimal health status.Only healthy clams were used for subsequent experiments.These conditions were carefully controlled to minimize any potential confounding factors that may affect the validity of the results.
We established four experimental groups, including the microplastic group (MMP), the microcystin-LR group (MMC), the microplastic_microcystin-LR group (MMP-MC), and one control group (Mcon), and selected Asian clams randomly for LC-MS experiments.The MC treatment concentration in the MMC group was set to 320 ug/L, and the MP treatment concentration in the MMP group was set to 640 mg/L.The treatment concentrations in the MMP-MC group were 320 ug/L (MC) and 640 mg/L (MP).Hepatopancreas samples were collected 96 hours after the challenge, and seven biological replicates from each treatment at each time point were used for LC-MS analysis (i.e., metabolomics).The foot, gill, and hepatopancreas samples were preserved in a 4% paraformaldehyde solution for histopathological analysis.

Metabolomics Analyses
All chemicals and solvents were analytical or LC-MS grade.Water, methanol, acetonitrile, and formic acid were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA).L-2-chlorophenylalanine was from Shanghai Heng Chuang Bio-technology Co., Ltd.(Shanghai, China).100 mg accurately weighed sample was transferred to a 1.5 mL Eppendorf tube.Two small steel balls were added to the tube.L-2-chlorophenylalanine (0.3 mg/mL) dissolved in methanol as internal standard and mixture of methanol and water (4/1, vol/vol) were added to each sample.Samples were stored at − 20 ℃ for 2 min and then grinded at 60 Hz for 2 min, and the whole samples were extracted by ultrasonic for 10 min in ice-water bath, stored at − 20 ℃ for 30 min.The extract was centrifuged at 4 °C (13,000 rpm) for 10 min.Supernatant in a glass vial was dried in a freeze concentration centrifugal dryer.The mixture of methanol and water (1/4, vol/vol) was added to each sample, samples vortexed for 30 s, extracted by ultrasonic for 3 min in icewater bat, and then placed at − 20 °C for 2 h.Samples were centrifuged at 4 °C (13,000 rpm) for 10 min.The supernatants from each tube were collected using crystal syringes, filtered through 0.22 μm microfilters, and transferred to LC vials.The vials were stored at − 80 °C until LC-MS analysis.QC samples were prepared by mixing aliquot of all samples to be a pooled sample.
Data acquisition was performed in full scan mode (m/z ranges from 50 to 1000) combined with MSE mode, including 2 independent scans with different collision energies (CE) alternatively acquired during the run.Parameters of mass spectrometry were as follows: a low-energy scan (CE 4 eV) and a high-energy scan (CE ramp 20-45 eV) to fragment the ions.Argon (99.999%) was used as collisioninduced dissociation gas; scan time, 0.2 s; interscan delay, 0.02s; capillary voltage, 2.5 kV; cone voltage, 40 V; source temperature, 115 °C; desolvation gas temperature, 450 °C; desolvation gas flow, 900 L/h.
The QCs were injected at regular intervals throughout the analytical run to provide a set of data from which repeatability can be assessed.

Data Preprocessing and Statistical Analysis
The original LC-MS data were processed by software Progenesis QI V2.3 (Nonlinear, Dynamics, Newcastle, UK) for baseline filtering, peak identification, integral, retention time correction, peak alignment, and normalization.Main parameters of 5 ppm precursor tolerance, 10 ppm product tolerance, and 5% product ion threshold were applied.Compound identifications were based on precise mass-to-charge ratio (M/z), secondary fragments, and isotopic distribution using The Human Metabolome Database (HMDB), Lipid maps (V2.3),Metlin, EMDB, PMDB, and self-built databases to do qualitative analysis.
The extracted data were then further processed by removing any peaks with a missing value (ion intensity = 0) in more than 50% in groups, by replacing zero value by half of the minimum value, and by screening according to the qualitative results of the compound.Compounds with resulting scores below 36 (out of 60) points were also deemed to be inaccurate and removed.A data matrix was combined from the positive and negative ion data.
The matrix was imported in R to carry out Principal Component Analysis (PCA) to observe the overall distribution among the samples and the stability of the whole analysis process.Orthogonal Partial Least-Squares-Discriminant Analysis (OPLS-DA) and Partial Least-Squares-Discriminant Analysis (PLS-DA) were utilized to distinguish the metabolites that differ between groups.To prevent overfitting, sevenfold cross-validation and 200 Response Permutation Testing (RPT) were used to evaluate the quality of the model.
Variable Importance of Projection (VIP) values obtained from the OPLS-DA model were used to rank the overall contribution of each variable to group discrimination.A two-tailed Student T-test was further used to verify whether the metabolites of difference between groups were significant.Differential metabolites were selected with VIP values greater than 1.0 and p values less than 0.05.

Histopathological Analysis
The foot, gill, and hepatopancreas of four clams from each group were fixed in 4% paraformaldehyde solution (Phygene, China).After being dehydrated and transparentized, tissues were embedded in paraffin wax and cut into 6-μm-thick sections.Then, the paraffin sections were stained with hematoxylin/eosin (H&E) and observed under a microscope (Olympus, Japan).

Effect of Microplastic and Microcystin-LR on the Survival of C. fluminea
As shown in Fig. 1, the 96 h LC50 value of microplastic in C. fluminea was calculated as 858.2 mg/L, and the 95% confidence interval was 840.5-875.7 mg/L.As shown in Fig. 2, the 96 h LC50 value of microcystin-LR in C. fluminea was calculated as 433.7 μg/L, and the 95% confidence interval was 424.6-442.7 mg/L.

Metabolic Responses of Hepatopancreas to Microplastic and Microcystin-LR Stress
In this experiment, six comparison groups were generated, including a control group (Mcontrol) and three experimental groups (MMP, MMC, and MMP-MC).Table 1 shows the specific number of differential metabolites and their  3) and OPLS-DA (Fig. 4), which revealed significant differences in metabolites between each comparison group.

Analysis of Different Metabolites
To show the relationship more intuitively between samples and the expression differences of metabolites between different samples, we conducted hierarchical clustering of all significantly different metabolites and the expression amounts of significantly different metabolites in the top 50 according to VIP sorting, and the results are shown below (Fig. 5).Six groups were defined, correctly reflecting MC, MP, and MP-MC effects (MMC, MMP, MMP-MC) and revealing subtle differences between biological replicates, which  possibly were due to natural differences among individuals.Among these metabolites, most of them were classified as "amino acids, peptides, and analogues," "carbohydrates and carbohydrate conjugates," and "fatty acids and conjugates," such as leucodelphinidin, phenylalanine, β-alanine, α-Dglucose-1,6-bisphosphate, and suberic acid.

Metabolic Pathway Analysis
The p value of a metabolic pathway was calculated to assess the significance of the enrichment of that pathway, and pathways that showed significant enrichment were selected for the bubble plot.In comparison to the Mcontrol group, all three experimental groups (MMP, MMC, and MMP-MC) exhibited significant differences (p < 0.05) in the purine metabolism pathway.Glycerophospholipid metabolism and mTOR signaling pathway were significantly different between MMC and MMP compared to the Mcontrol group.Aminoacyl tRNA biosynthesis and phenylalanine, tyrosine, and tryptophan biosynthesis pathways were significantly different between MMP and MMP-MC compared to the Mcontrol group (Fig. 6).Significant (p < 0.05) differences were observed between MMP and MMC in multiple metabolic pathways including aminoacyl tRNA biosynthesis, D-amino acid metabolism, glycerophospholipid metabolism, and sulfur metabolism.Glycerophospholipid metabolism, oxidative phosphorylation, riboflavin metabolism, alanine, aspartate and glutamate metabolism, arachidonic acid metabolism, autophagy-other, taurine and hypotaurine metabolism, and ABC transporters pathways were significantly different between MMP and MMP-MC.Furthermore, significant differences were observed between MMC and MMP-MC in glycerophospholipid metabolism, FOXO signaling pathway, oxidative phosphorylation, aminoacyl tRNA biosynthesis, D-amino acid metabolism, glycine, serine, and threonine metabolism, and lysine biosynthesis pathway (Fig. 6).

Histopathological Index Observations
The foot tissues of C. fluminea exhibited dense sheet-like cells in both the control and all treatments (Fig. 8).The hepatopancreas of normal C. fluminea (control group) mainly consisted of the digestive tubule and connective tissue, with small or closed lumens of the digestive tubule Fig. 3 PCA plot for each comparison group.Note: the projected score value of each sample on the plane composed of the first principal component and the second principal component is the spatial coordinates, which can visually reflect the similarity or difference between samples.The elliptical region represents a 95% confidence interval and aligned epithelial and digestive cells.In MMP groups, enlargement of the lumen was observed in the hepatopancreas of C. fluminea in the high concentration treatment, with irregularly arranged and even exfoliated epithelial and digestive cells and vacuolation after exposure to high concentration (Fig. 9).Similarly, in MMC groups, enlargement of the lumen was observed in the hepatopancreas of C. fluminea in the high concentration treatment, with irregularly arranged and even exfoliated epithelial and digestive cells and vacuolation occurring in medium and high concentration treatments (Fig. 9).In normal gill tissues, the lymphatic vessels exhibited an orderly arrangement and kelp-like shape, with pyknotic cilia present on both sides of the ends of the lymphatic vessels (Fig. 9).The cilia of the epithelial cells in gills underwent slight and moderate cilia degeneration in the low (MC 40 μg/L; MC 80 μg/L; MP 80 mg/L; MP 160 mg/L) and medium (MC 160 μg/L; MP 320 mg/L) concentration microplastic treatments, while OPLS-DA maximizes between-group differences on t1, so it can directly distinguish between-group variation from t1, while orthogonal principal components reflect within-group variation severe cilia degeneration and corrosion were observed in the high concentration treatment (MC 320 μg/L; MP 640 mg/L) (Fig. 10).

Discussion
At present, microplastics and microcystic algal toxins are research hotspots in ecology and breeding (Zhang et al. 2019b;Pannetier et al. 2020).Asian clams are considered a potential ecological indicator biological and economic species, and the filter feeding behavior of shellfish also enriches higher concentrations of microplastics and microcystic algal toxins (Wang et al. 2018).Studying the metabolism of Asian clams could play an important role in understanding their metabolic mechanisms in shellfish, and it may be possible to find new ways to use biological means to remove microplastics and microcystic algal toxins from water.
In this study, we experimented with Asian clams that were in good health in the same farming environment.To avoid the stress caused by environmental changes, a oneweek temporary rearing was carried out before the official start of the experiment, during which bait was appropriately fed to ensure the immunity of the river clams.During the experiment, we could clearly observe that the microplastics group, the microcystic algal toxin group, and the microplastic-microcystic algal toxin composite group all had different degrees of mucus secretion, but no death was observed.

Histopathological Analysis
Gills are the major channel for material exchange in bivalves and are the principal targets of external contaminations (Teh et al. 2000).In this study, cilia degeneration and corrosion appeared in MC and NP treatments, particularly in high concentration treatment, which may be caused by oxidative damage (Zhang et al. 2020).Microplastics were ingested and filtered by the anterior cilia, which might have directly injured cilia and epithelial cells and caused high oxidative damage in these areas.Ciliary degeneration caused by oxidative damage is the most common tissue damage phenotype in C. fluminea (Wang et al. 2021).A marine bivalve (Mytilus galloprovincialis) was also observed to suffer ciliary degeneration and epithelial cell dysfunction following exposure to environmental pollutants (Yavaşoğlu et al. 2016).Microcystin are toxic metabolites produced by Microcystis, Anabaena, and other algae, which are commonly found in freshwater aquaculture ponds.It mainly targets the liver, destroys the structure of aquatic animal hepatocytes, causes hepatocyte deformation, and leads to liver dysfunction and has a time concentration dual effect (Martins et al. 2019).In this experiment, the lumen expansion of the hepatopancreas was observed in each treatment group.In the high concentration group, epithelial cells and digestive cells were arranged irregularly and even exfoliated.The purines are a group of molecules used by all cells for many vital biochemical processes including energyrequiring enzymatic reactions, cofactor-requiring reactions, synthesis of DNA or RNA, signaling pathways within and between cells, and other processes.Defects in some of the enzymes of purine metabolism are known to be associated with specific clinical disorders, and neurological problems may be a presenting sign or the predominant clinical problem for several of them.Hypoxanthine is an essential metabolite used to degrade adenine nucleotide that accumulates in biological tissues (Miller et al. 2020).It can be catalyzed and converted to xanthine and then to uric acid to produce excessive reactive oxygen species (ROS) and hydrogen peroxide (Al-Shehri et al. 2020).The negative regulation of hypoxanthine is likely related to xanthine oxidase (XO)-mediated innate immune response in clam after MP and MC treatment.

Analysis of Common Differential Metabolite and Metabolic Pathways
The phenylalanine, tyrosine, and tryptophan biosynthesis pathway showed significant (p < 0.05) changes in the MMP-MC/Mcontrol and MMP/MMC treatment groups, while no significant (p < 0.05) changes were observed in the MC treatment group alone.These findings suggest that only MC treatment alone would not have a significant (p < 0.05) impact on this pathway.Furthermore, neither MC treatment alone nor MP treatment alone had any effect on L-phenylalanine.However, the MP-MC combined treatment induced the negative regulation of L-phenylalanine, which may imply that this combination treatment could expand the toxicity of pollutants.In the phenylalanine, tyrosine, and tryptophan biosynthesis pathway, phenylalanine is a metabolite serving as a precursor of tyrosine that is transformed into catecholamine neurotransmitters (Flydal and Martinez 2013).The dysregulation of the phenylalanine, tyrosine, and tryptophan biosynthesis implied that microplastic and microcystin-LR were toxic to the central nervous system (CNS) (Blaise et al. 2017).The negative regulation of phenylalanine might result in the dysregulation of other amino acids, such as tryptophan and tyrosine in the CNS, because phenylalanine competes with other neutral amino acids (Clarke et al. 2016).The negative regulation of tryptophan in the Asian clam hepatopancreas exposed to the microplastic and microcystin-LR mixture might be associated with the negative regulation of the neurotransmitter serotonin, as it can compensate for the damage caused by a dopaminergic system (Sokolowska-Mikolajczyk et al. 2015).Previous studies have shown that in mice, the effect of prolonging span can be achieved by activating the metabolic axis of glycine, serine, and threonine metabolism to enhance detoxification, maintain molecular synthesis turnover, repair/maintenance, reduce oxidative stress, and maintain liver function.We speculate that this pathway may play a similar role in bivalves (Aon et al. 2020).
Compared with the control group, we observed a reduction in the levels of many kinds of fatty acids in the hepatopancreas of treated clams, including arachidonic acid, eicosapentaenoic acid, elaidic acid, adrenic acid, and eicosadienoic acid.Fatty acids play a crucial role as sources of energy and are essential components of cellular membranes.Moreover, they have been shown to modulate immune responses (Fritsche 2006).Stress and disease are known to be high energy-demanding processes, and various studies have explored the impact of biotic and abiotic factors on the composition and amount of fatty acids in mollusks (Zhukova 2019).For instance, in Mytilus galloprovincialis clams exposed to cadmium and copper and polycyclic aromatic hydrocarbon contamination, a reduction in polyunsaturated fatty acid (PUFA) and non-methylene-interrupted fatty acid content has been observed (Fokina et al. 2013) (Signa et al. 2015).Similarly, under salinity stress, a decrease in PUFA content was observed in cockles (Cerastoderma edule) (Gonçalves et al. 2017).In response to pathogens, several studies have reported a decrease in various fatty acids in the hemolymph of Vibrio-challenged P. canaliculus (Nguyen et al. 2018;Van Nguyen and Alfaro 2019).Therefore, the observed decrease in fatty acid levels in the hepatopancreas samples of treated clams may indicate high energy demands for metabolic and immune responses during the treatment.

Analysis of Distinct Differential Metabolite and Metabolic Pathways
MMP has a number of metabolites associated with inflammatory regulation that are less expressed than MMC, such as 3,4-dihydro-2H-1-benzopyran-2-one, 2-hydroxycinnamic acid, 4-oxo-2-nonenal, and docosapentaenoic acid (22n-6), which may suggest that MC can provoke a more severe inflammatory response to river clams than MP.Levels of lipid and glucose metabolism-related metabolites such as D-fructose, 3-hydroxyeicosanoylcarnitine, and FAD also increased in MMC.The energy required for ion and osmotic regulation comes mainly from carbohydrate metabolism (Jiang et al. 2019).Similarly, lipids have energy storage and energy supply functions in organisms as well as a variety of functions in structures and biological processes.The previous study showed that lipid metabolism of Scophthalmus maximus was changed under osmotic stress, including fat digestion and absorption, cholesterol metabolism (Liu et al. 2021).Thus, levels of these metabolites were high in the MMC group because Asian clams required more energy for MC compared to the NP.We also observed significant differences in amino acid biosynthetic pathways, such as aminoacyl-tRNA biosynthesis, D-amino acid metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, and lysine biosynthesis, between the MMP/MMC group.Alanine and phenylalanine are essential intermediates in the tricarboxylic acid cycle (TCA cycle) and play important roles in the organism, such as carbohydrate and lipid metabolism (Xu et al. 2012;Li et al. 2017b;Mishra et al. 2017;Liu et al. 2021).Increases in glycine and proline levels during environmental stress adaptation were also observed in other bivalves (Chen et al. 2023).Isoleucine is a amino acid and is involved in protein synthesis, energy production, neurotransmission, and immunity (Zhang et al. 2017).Alanine and proline are also important organic buffering agents and antioxidants (Saum et al. 2013).These results suggest that amino acid metabolism may be a key factor distinguishing Asian clams' resistance to MP from their resistance to MC.
In both the comparisons between MMP-MC and MMP groups, as well as between MMP-MC and MMC groups, higher expression levels of lipid metabolism-related metabolites, glycerophosphocholine and glycerophosphoinositol, were observed.They play crucial roles in cell membrane composition and cellular signal transduction while also exhibiting antioxidative properties and regulating cellular physiological functions (Zhou et al. 2018;Xie et al. 2020).Glycerophosphocholine is a major component of cell membranes, along with phosphatidylcholine, forming the phospholipid bilayer that maintains membrane integrity and stability (Fernández-Murray and McMaster 2005).It exhibits antioxidative activity, neutralizing intracellular free radicals and protecting cells from oxidative damage.On the other hand, glycerophosphoinositol is a key component in cell signal transduction, generating various secondary signaling molecules through different enzymecatalyzed reactions to regulate cellular processes such as proliferation, differentiation, and apoptosis (Corda et al. 2009).Additionally, glycerophosphoinositol participates in regulating cell survival and physiological stress responses, significantly influencing cell viability and adaptability.The higher expression of glycerophosphocholine and glycerophosphoinositol may indicate that the combined exposure of MP-MC enhances toxicity.

Conclusion
This study described, for the first time, metabolic disruptions in molecular pathways in Asian clam tissues as a response to the microplastics and microcystin-LR stress.These pathways are mostly involved in energy metabolism, oxidative stress, amino acid metabolism, fatty acid metabolism, signaling, and amino acid biosynthesis pathway processes.The response of C. fluminea to combined exposure of MP and MC requires substantial utilization of fatty acids, and the co-exposure of MP and MC may potentiate the toxicity of pollutants.While this study reports on samples collected in a single timing, further sampling across time points may provide further information regarding the development of stress responses as well as an indication of susceptibility and resilience patterns among Asian clams.For a more comprehensive investigation of the complex relationship between species and contaminants, a multiplatform and multiomics (e.g., genomics, transcriptomics, proteomics, and metabolomics) approach is suggested.
upregulated and downregulated status.The MMC/Mcontrol comparison group had the highest number of differential metabolites, with a total of 564, while the MMC/MMP comparison group had the lowest number, with 258.All comparison groups were ranked in descending order based on the total number of differential metabolites: MMC/Mcontrol > MMP-MC/Mcontrol > MMP/Mcontrol > MMP-MC/ MM > MMP-MC/MMP > MMC/MMP.The experimental groups were analyzed using PCA (Fig.

Fig. 1 Fig. 2
Fig. 1 Effects of different concentrations of microplastic on survival rate at 96 h

Fig. 4
Fig. 4 OPLS-DA plot for each comparison group.Note: on the OPLS-DA score plot, there are two principal components, the predicted principal component and the orthogonal principal component.

Fig. 5 Fig. 6
Fig.5Heatmap for each comparison group.The abscissa represents the sample name, and the ordinate represents the differential metabolite.Color from blue to red indicates that the expression abundance of metabolites is low to high, that is, the redder indicates the higher the expression abundance of differential metabolites ◂

Fig. 7 Fig. 8
Fig.7Correlation plot between each comparison group.Red indicates a positive correlation and blue indicates a negative correlation.The larger the dot, the greater the correlation coefficient between the two variables.We performed a correlation analysis for the top 20 significantly different metabolites sorted by VIP ◂

Table 1
The number of differential metabolites and their upward and downward regulation